Regulation of protamine gene expression in an in vitro homologous system.

An in vitro transcription system from the trout testis nuclei was developed to study trout protamine gene expression. The protamine promoter contains, among others, two regulatory elements: 1) a cAMP-responsive element or CRE element (TGACGTCA) which is present in position 5' to TATA box, and 2) GC box (CCGCCC) which is present in position 3' to TATA box. The removal of the CRE-binding protein by titration (by the addition of appropriate oligonucleotides to the incubation mixture) resulted in a decrease in transcription of the protamine gene. These results were confirmed by experiments in which the pure CRE-binding factor (TPBP1) was used, as well as by those where a stimulatory effect of cAMP on protamine promoter transcription was observed. On the other hand, addition of oligonucleotides containing the GC-box sequence enhanced the protamine gene transcription indicating that the protein (Sp1 like) which binds to this sequence acts as a repressor of protamine gene expression. These results confirm the previously proposed model which suggested that the GC box played a role in negative regulation of the protamine gene expression. Involvement of some other factors in this process was also discussed.

Cloning of two cDNAs encoding calnexin-like and calreticulin-like proteins from maize (Zea mays) leaves: identification of potential calcium-binding domains.

Two cDNAs encoding calnexin (Cln)-like and calreticulin (Crl)-like proteins have been isolated by immunoscreening of a maize leaf cDNA library. In the deduced amino acid (aa) sequences, several regions that are conserved for Cln and Crl proteins from all sources have been identified. These regions can be classified into two distinct motifs which are repeated four times each in Cln and three times each in Crl sequences. One of these motifs, containing a highly acidic 17-aa sequence, has high homology to a Ca(2+)-binding domain previously characterized in both Cln and Crl from mammalian tissues. Motifs for retention in endoplasmic reticulum (Crl) and for an integral membrane-spanning sequence (Cln) have also been identified.

DNA polymerases of rabbit mammary gland: partial purification, characterization and changes in DNA polymerase activities as a function of physiological state.

DNA polymerases alpha and beta were partially purified from rabbit mammary gland, and their properties were examined. Many of these properties (sedimentation coefficients, pH optima, divalent cation requirements, sensitivity to various inhibitors) are similar to those commonly regarded as typical of eukaryotic DNA polymerases alpha and beta. Effect of stage of pregnancy and lactation on the levels of activity of DNA polymerases alpha, beta and gamma in rabbit mammary gland was examined. These experiments reveal a considerable variation of DNA polymerase-alpha activity; the highest enzyme activity is observed on day-20 of pregnancy and on day-1 of lactation.

Evidence for the selective replication of dwarf pea DNA evoked by exogenous gibberellin application.

Characteristics of the total DNA preparations isolated from apical parts of dwarf pea seedlings untreated and treated with gibberellic acid (GA3) were compared. Analytical centrifugation in a self-generated CsCl density gradient revealed the occurrence of a heavy satellite DNA band (p = 1.712 g X cm-3) in addition to the main DNA band (p = 1.696 g X cm-3) in the DNA preparation extracted from GA3-treated seedlings, that could not be detected in the DNA isolated from untreated plants. The existence of this GC-rich DNA fraction was additionally confirmed by means of derivative DNA melting profiles. Comparison of the reassociation kinetics obtained for control DNA with DNA from GA3-treated plants showed changes in the percentage distribution of three main DNA sequence classes, with different repetition frequency in the haploid pea genome. It is postulated that such a variation in the percentage of different C0t families might reflect the selective DNA replication evoked by hormonal treatment of dwarf pea plants.

Preferential stimulation of the plant mRNA synthesis by gibberellic acid.

1. Treatment of the etiolated maize seedlings with the plant hormone, gibberellic acid results in a significant enhancement of heavy polyribosome formation. 2. This is accompanied by highly increased incorporation of the labelled RNA precursors into RNA engaged in the polyribosomal complex, as well as by an increased rate of protein synthesis in vivo. 3. Determination of the specific radioactivity of particular RNA classes isolated from polyribosomes reveals that gibberellic acid stimulates mostly the synthesis of the rapidly labelled, non-ribosomal RNA fraction. 4. A considerable amount of this rapidly labelled RNA fraction, whose synthesis is preferentially stimulated by exogenous gibberellic acid contains poly(A) sequences, as shown by affinity chromatography on oligo (dT)-cellulose indicating that phytohormone causes an increased transcription of mRNA in etiolated maize seedlings. 5. When [3H]adenosine served as the RNA precursor it was found that the ratio between the heteropolymeric and polyadenylic parts of the poly(A)-RNA chain markedly changed under gibberellin treatment, suggesting that, in addition to an increased rate of mRNA synthesis, the plant hormone also affects the process of post-transcriptional polyadenylation of the newly made mRNA precursors. Possible extension of the polyadenylate segment in the presence of gibberellin may account for a longer functional half-life of the mRNA synthesized in plants treated with the phytohormone, and may explain significantly enhanced heavy polyribosome formation, as well as a higher efficiency of protein synthesis in plants treated with gibberellic acid.